Bacterial infections continue to be one of the major cause of diseases inflicting infants and children, particularly in developing countries (Osrin, David et al. (2004) Current Opinion in Infectious Diseases 17(3): 217-224; Thayer, Durrane, and Zaidi, Anita K. M. (2009) Pediatric Infectious Disease Journal 28(1): S3-S9; Sáez-Llorens, Xavier et al. 2003 Lancet 361(9375): 2139-2148; Thapar, Nikhil et al. 2004 Lancet 363(9409): 641-653). The most common pathogens are Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis (Pollard, Andrew J. et al. (2009) Nature Reviews Immunology 9(3): 213-220), Staphylococcus aureus, Shigella, Salmonella, Vibrio cholerae etc. A large number of children die each year as a result of such infections.
Polysaccharide antigens have been one of the major components of bacterial vaccines used to prevent diseases associated with Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae (Larry K. Pickering et al. (1985) Infectious Diseases Newsletter 4(11): 84-87), and Salmonella enterica serovar Typhi (Hessel L et al. (1999) Eur J Clin Microbiol & Infect Dis., 18(9): 609-620). However, most of the bacterial polysaccharides are T-cell independent antigens preventing development of memory B-cells leading to poor immune response to such antigens in children below two years and elderly persons. Covalent conjugation of polysaccharide antigens to protein carrier bestows them the ability to generate humoral response, and impart them the capabilities of T-cell dependent antigens. Such conjugates have been proven to be efficient in preventing diseases caused by bacterial pathogens. Polysaccharide conjugate vaccines have been licensed for use in many parts of the world for more than two decades (Adams, William G. et al. (1993) JAMA 269(2): 221-226).
Purification of polysaccharide protein conjugates has always been a challenge. Such conjugates are known to be associated with contaminants such as un-reacted polysaccharide (free polysaccharide), un-reacted carrier protein (free protein), low molecular weight conjugates, and other chemicals used for affecting conjugation such as linkers, coupling agents etc. Such contaminants are highly undesirable in a product which is intended for use as a vaccine. For example, a high amount of free polysaccharide is undesirable in a vaccine composition as it might interfere with the immunological function of the conjugate (Peeters, C. A. M. et al. (1992) Vaccine 10(12): 833-840). The contaminants often differ in molecular size, ionic charges and hydrophobicity making it difficult to employ single chromatography step to achieve the purity levels desired for it to be used as vaccine.
Polysaccharide protein conjugates have often been purified from impurities or contaminants by various standard techniques such as density gradient centrifugation, ultrafiltration with ammonium sulfate fractionation (U.S. Pat. No. 6,146,902), ethanol precipitation, gel filtration or size exclusion chromatography, hydrophobic interaction chromatography or ion exchange chromatography.
Ion exchange chromatography has been mostly found suitable to purify the polysaccharides, but it has not been found appropriate to purify conjugates and more particularly when the conjugates are to be purified on a larger scale. Simon, Raphael (WO2012061400) describes a process for polysaccharide protein conjugate purification by binding the conjugate to ion exchange matrix and eluting it subsequently to obtain the purified conjugate. Such processes also results in binding of the free polysaccharide as the conjugate often exhibit charges similar to the free polysaccharide making the purification of conjugate difficult.
Polysaccharide protein conjugates may also be purified using an adsorption method based on hydrophobicity, which will adsorb the conjugate but not the free polysaccharide as the latter tends to be less hydrophobic, by using a high concentration of salt (Lees, Andrew et al. WO2011017101; Pawlowski, Andrzeg (2000) Vaccine 18: 1873-1885). Such purification is achieved by exploiting the hydrophobic nature of the protein which is mainly governed by the proportion of the non-polar surface areas of the protein as well as their spatial distribution. If the conjugated protein is less hydrophobic, hydrophobic interaction chromatography may not be a preferred choice of purifying such conjugates.
Gel filtration chromatography has been most commonly used to purify polysaccharide protein conjugates (Lees, Andrew et al. (1996) Vaccine 14(3): 190-198; Libon, Christine (2002) Vaccine 20: 2174-2180; Jennings, H. J. and Lugòwski, C. (1981) J. Immunology 127: 1011-1018). However such techniques suffer from several limitations. For example, purification using molecular sieving by gel filtration chromatography could only be achieved by sacrificing yields due to inadequate resolution of crude conjugate. Because of the narrow fractionation range, pooling of appropriate fraction containing the desired conjugate requires considerable skills. Any error in collecting fraction could lead to significant loss of the conjugate or increases the risk of association of contaminants with the conjugate as the conjugate and contaminants separate at a very narrow range (see FIG. 1a). Further, it requires large amount of gel filtration matrix, and significantly increased process time, in addition to the difficulties associated with column packing. All these factors lead to higher cost of production which makes the vaccines unaffordable, limiting their wider use in vaccination programs.
The complexity associated with conjugation process usually results in highly heterogeneous array of contaminants often differing in physical and chemical properties such as molecular size, ionic charges, hydrophobicity etc. These factors have made the separation based on single principle of chromatography inadequate to achieve the required degree of purification for the conjugate to be used as vaccine. Therefore, sometimes more than one chromatography steps have been used to purify polysaccharide protein conjugates. Fattom, Ali et al. (Infection and Immunity (1988) 56(9): 2292-2298) describes a two step process of purifying a polysaccharide protein conjugate wherein the conjugate is partially purified over a gel filtration matrix followed by capturing onto a hydrophobic medium to obtain the purified conjugate.
Such methods compromises overall yield of the conjugate due to multiple chromatography steps and also results in increased process time.
Thus production of significant quantities of polysaccharide protein conjugates for use in vaccines has been hindered due to complexities in the prior art processes leading to low yields and high cost of production. Therefore, there is a need to develop alternate method of purification of polysaccharide protein conjugates which offers ease of manufacture, which is less time consuming, and at the same time offering good yields. The present invention provides an unexpectedly efficient method of removing impurities or contaminants from polysaccharide protein conjugates, employing mixed mode chromatography (Orlovsky, Vlad et al (2011) Chromatography Today, 4(3) 24-28; WO2005082483; WO2009131526, WO2010005364). This surprisingly effective method addresses the long-standing problems associated with the prior art processes used to purify polysaccharide protein conjugates. The process of the invention is operationally simple, easily scalable, requires fewer resources, and offers greater yields and product of consistent quality.